United States Patent [19]
`Moore et al.
`
`[54]
`
`[75]
`
`HYBRID DNA PREPARED BINDING
`COMPOSITION
`
`Inventors: Kevin W. Moore, San Bruno;
`Alejandro Zaffaroni, Atherton, both of
`Calif.
`
`[73]
`
`Assignee: Schering Corporation
`
`[21]
`
`Appl. No.: 461,071
`
`[22]
`
`Fikd:
`
`Jun. 5, 1995
`
`[62]
`
`[51]
`
`[52]
`[58]
`
`[56]
`
`Related U.S. Application Data
`
`Division of Ser. No. 394,923, Feb. 23, 1995, abandoned,
`which is a continuation of Ser. No. 210,540, Mar. 17, 1994,
`abandoned, which i> a continuation of Ser. No. 61,760, May
`13, 19~X\ abandoned, which is a continuation of Ser. NO.
`928,526, Aug. 11, 1992, abandoned, which is a continuation
`of Ser. No. 740,862, Jul. 31, 1991, abandoned, which is a
`continuation of Ser. No. 235,835, Aug. 18, 1988, abandoned,
`which is a continuation of Ser. No. 55S,551, Dec. 5, 19S3,
`Pat. No. 4,642,334, which is a continuation of Ser. No.
`358,414, Mar. 15, 1982, abandoned.
`Int. Cl.6
`CUP 21/02; C12P 21/08;
`C12N 1/21; C12N 15/13
`U.S. Cl . .................. 435/696; 435/172.3; 435/252.33
`Field of Search .............................. 73'i/240.2, 172.3,
`735/69.1, 69.6; 514/2; 530/387.1, 387.3;
`435/69.1, 69.6, 172.1, 172.3, 320.1, 252.33
`
`......................
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`
`4,036,945
`4,642,334
`
`7! 1977 Haber ct al.
`424/1.49
`2/1987 Moore et al. ...... .... .......... ...... 530/388
`
`FOREIGN PATENT DOCUMENTS
`
`AU U35 265
`
`9/19S1 European Pat. Off ..
`
`01HER PUBLICATIONS
`
`Bodty t! al., "Human Canctr Dt!tc!ion and Immuno!htrapy
`with Conjugated and Non-conjugattd Monoclonal Antibod(cid:173)
`ies", Anticancer Res., vol. 16, pp. 661-674 (1996).
`Jain, "Vascular and Interstitral Earners to Delivery of Thera(cid:173)
`peutic Agents in Tumors", Cancer Metastasis Rev. vol. 9(3),
`pp. 253-266, 1990 (Abstract only).
`Baumgartner et a!., "Immunotherapy of Endothoxemia and
`Stplicemia", Immunobiol., vol. 187, pp. 464-477, (1993).
`Givol, 1974, Jissays in Biochemistry 10:73-104.
`Gough, Aug., 1981, TIBS 203-205.
`Early et al., 1981, Genetic Engineering: Principles and
`Methods 3:157-183.
`Leder, 1982, Scientific American 19:2702-2710.
`
`111111111111111111111111111111111111111111111111111111111111111111111111111
`US005840545A
`[11] Patent Number:
`[45] Date of Patent:
`
`5,840,545
`Nov. 24, 1998
`
`Gough ct al., 1980, Biochemistly 19: 2702-2710.
`Adams et al., 1980, Biochemi5lry 19: 2711-2719.
`Inhar et al., Proc. Nat. i\cad. Sci. USi\ 69(9):2659-2662.
`Hochman et al., 1973, Biochemistry 12(6):1130--1135.
`Sharon eta!., 1976, Biochemi5try 15(7):1591-1594.
`Kooistra et al., 1978, Biochemistry 17(2):345-351.
`Accolla et al., 1980, Proc. Nat. Acad. Sci., USA.
`77(1):563-566.
`O'Sullivan et al., 1979, Annals of Clinical Biochemistry
`16:221-240.
`Miozzari <::tal., 1978, J. Bacterio/133(3):1457-1466.
`Pluckthun, 1991, Bio/technology 9:545-551.
`Kakimoto et al., 1974, The Journal of Immunology
`112(4): 1373-1382.
`al., 1978, Proc. Nat/. Acad. Sci. USA.
`Lin
`et
`75(6):2649-2653.
`Rosemhlatt et al., 1978, Riochemistrv 17:3877-3882.
`Cunningham, Understanding lmmun~logy, pp. 38-39.
`Alberts, 1989,Molecular biology of the cell, pp. 1025-1026.
`Francis et al., 1974, Proc. Nat.llcad. Sci., 71:1123-1127.
`Journal
`of
`Immunology
`1980,
`Raso
`et
`al.,
`125(6):2610-2616.
`Hales et a!., 1980, Merhods in enzymology 70:334-355.
`O'Sullivan et al., 1981, Methods in enzymology 73:147-166.
`Boss eta!., 1984, Nucleic Acids Research 12:3791-3806.
`Wood eta!., 1985, Nature 314:446-448.
`Cabilly et al., 1984, Proc. Nat/.
`/\cad. Sci. USA.
`81:3273-3277.
`Kenton et al., 1984, Proc. Nat/.
`81:2955-2599.
`Liu et al., 1984, Proc. Nat/. Acad. Sci. USA 81:5369-5373.
`Gherna et al., 1985, American Type Culture Collection
`Sixteenth edition, p. 243.
`Gubler et al., 1986, J. Immunol. 136:2492-2497.
`Saxena et al., 1970, Biochemistry 9:5015-5023.
`
`/\cad. Sci. USA.
`
`Primary lixaminer--David Guzo
`Attorney, Agent, or Firm-Cynthia L. Foulk<::; Edwin P.
`Ching
`
`[57]
`
`ABSTRACT
`
`Proteinaceous binding compositions are prepared employing
`hybrid DNA technology, where the variable region polypep(cid:173)
`tides of immunoglobulins arc substantially reproduced to
`provide relatively small protein molecules having binding
`specificity and lacking the undesirable aspects of the heavy
`rtgions of immunoglobulins. Tht compositions find a widt
`range of use, particularly for physiological purposes for
`diagnosis and therapy. TI1e binding compositions may be
`modified by labeling with radioisotopes, fluorescers, and
`toxins for specific applications in diagnosis or therapy.
`
`2 Claims, No Drawings
`
`Sanofi/Regeneron Ex. 1019, pg 577
`
`Merck Ex. 1019, pg 603
`
`

`
`5,840,545
`
`1
`HYBRID DNA PREPARED BINDING
`COMPOSITION
`
`This application is a division of application Ser. No.
`08/394,923, filed Feb. 23, 1995, now abandoned, which is a
`continuation of application Ser. No. 08/210,540, filed Mar.
`17, 1994, now abandoned, which is a continuation of appli(cid:173)
`cation Ser. No. 08/061,760, filed May 13, 1993, now
`abandoned, which is a continuation of application Ser. No.
`07/928,526, filed Aug. 11, 1992, now abandoned, which is 10
`a continuation of application Ser. No. 07/740,862, filed Jul.
`31, 1991, now abandoned, which is a continuation of appli(cid:173)
`cation Ser. No. 07/235,835, filed Aug. 18, 1988, now
`abandoned, which is a continuation of application Ser. No.
`06/558,551, filed Dec. 5, 1983, now U.S. Pat. No. 4,642,334, 15
`which is a continuation of application Ser. No. Oli/358,414,
`filed Mar. 15, 1982, now abandoned.
`
`BACKGROUND OF THE INVENTION
`
`20
`
`2
`host antibodies against the constant region of the immuno(cid:173)
`globulin or against any other part of the molecule.
`It is therefore important that methods be developed which
`permit the preparation of homogeneous compositions hav(cid:173)
`ing high specificity for a particular ligand, while avoiding
`the shortcomings of complete immunoglobulins, and pro(cid:173)
`viding the many advantages of lower molecular weight.
`2. Description of the Prior Art
`Discussions concerning variable regions of heavy and
`light chains of immunoglobulins may be found in Sharon
`and Givol, Biochem. (1976) 15:1591-1594; Rosemblatt and
`Haber, Biochem. (1978) 17:3877-3882; and Early and
`Hood, Genetic Engineering (1981) 3:157-188. Synthesis of
`part of a mouse immunoglobulin light chain in a bacterial
`clone is described by Amster et al., Nucleic Acids Res.
`(1980) 8:2055-2065. Sec also the references cited through(cid:173)
`out the specification concerning particular methodologies
`and compositions.
`
`SUMMARY OP TilE INVENTION
`
`Novel protein complexes are provided by producing
`homogeneous compositions defining the variable regions of
`the light and heavy chains of an immunoglobulin, which
`individually or together fonn a specific binding complex to
`a predetermined haptenic or determinant site. Employing
`hybrid DNA technology, eDNA is tailored to remove nucle(cid:173)
`otides extranem1s to the variable regions of the light and
`heavy chains. The resulting tailored ds eDNA is inserted into
`an appropriate expression vector which is then introduced
`into a host for transcription and translation. The resulting
`truncated light and heavy chains define at least a major
`portion of the variable regions and are combined to form a
`complex capable of specifically binding to a predetermined
`35 haptenic site with high affinity.
`
`30
`
`DESCRIPTION OF THE SPECIFIC
`EMBODIMENTS
`
`40
`
`1. Field of the Invention
`The mammalian immunological system is unique in its
`broad ability to produce protein com pounds having
`extremely high specificity for a particular molecular struc(cid:173)
`ture. That is, the proteins or immunoglobulins which arc
`produced have a conformation which is specifically ahle to 25
`complement a particular structure, so that binding occurs
`with high affinity. In this manner, the mammalian immune
`system is able to respond to invasions of foreign molecules,
`particularly proteins in surface membranes of
`microorganisms, and toxins, resulting in detoxification or
`destruction of the invader, without adverse effects on the
`host.
`The primary immunoglobulin involved in the defensive
`mechanism is gamma-globulin (IgG). This immunoglobulin,
`which is a glycoprotein of about 150,000 daltons, has four
`chains, two heavy chains and two light chains. Each of the
`chains has a variable region and a constant region. The
`variable regions are concerned with the binding specificity
`of !he immunoglobulin, while the constant regions have a
`number of other functions which do not directly relate to the
`antibody affinity.
`In many situations it would he desirable to have mol(cid:173)
`ecules which are substantially smaller than the
`immunoglobulins, while still providing the specificity and
`affinity which the immunoglobulins afford. Smaller mol(cid:173)
`ecules can provide for shorter residence times in a mamma(cid:173)
`lian host. In addition, where the immunoglobulin has to be
`bound to another molecule, it will be frequently desirable to
`minimize the size of the final product. Also there are many
`economies in being able to produce a smaller molecule
`which fullllls the function of a larger molecule.
`There arc situations where it will be desirable to be able
`to have a large number of molecules compactly held
`together. By having smaller molecules, a greater number can
`be brought together into a smaller space. Furthermore,
`where the binding molecule can be prepared by hybrid UNA
`technology, one has the opportunity to bind the binding
`portion of the molecule to a wide variety of other
`polypeptides, so that one can have the binding molecule
`covalently bonded at one or both ends to a polypeptide
`chain.
`Where in1munoglobulins are used in in vivo diagnosis or
`therapy, antisera from an allogenic host or from a mono(cid:173)
`clonal antibody may be immunogenic. Purthermore, when 65
`conjugates of other molecules to the antibody are employed,
`the resulting conjugate may become immunogenic and elicit
`
`45
`
`50
`
`5S
`
`The subject invention concerns a hybrid DNA strategy for
`the preparation of specific binding polypeptides, normally
`comprised of two different polypeptide chains, which
`together assume a conformation having high binding affinity
`to a predetermined ligand or haptenic site thereof. The
`polypeptide chains form binding sites which specifically
`hind to a predetermined ligand to form a complex having
`strong binding between the ligand and the binding site. The
`binding constant or avidity will generally be greater than
`, more usually greater than 106
`105
`, and preferably greater
`than 108 . The haptenic binding site or determinant binding
`site of the polypeptide chain may be associated with a hapten
`or antigen.
`One or both of the different polypeptide chains derived
`from the variable region of !he light and heavy chains of an
`immunoglobulin may be used Lo provide specilic binding to
`a ligand. For the most part each of the polypeptide chains of
`the light and heavy variable regions would he employed
`together for binding to the ligand. In describing this
`invention, it will be understood that while the two different
`6u chains are indicated as forming a complex, either of the
`chains could he used individually, where feasible due to
`sullicien! binding allini!y of the particular chain to the
`reciprocal ligand.
`The two polypeptide chains which, individually or
`together, provide the compositions of this invention will
`form a receptor site, analogous to the binding site of an
`immunoglobulin. The composition will be referred to as an
`
`Sanofi/Regeneron Ex. 1019, pg 578
`
`Merck Ex. 1019, pg 604
`
`

`
`5,840,545
`
`15
`
`3
`rFv with the individual chains referred to as L-rFv or H-rFv.
`The L- and II- designations will normally mean light and
`heavy respectively, but in some instances the two chains
`may be the same and derived from either the light or heavy
`chain sequences. The polypeptide chains of the rFv will
`generally have fewer than 125 amino acids, more usually
`fewer than about 120 an1ino acids, while normally having
`greater than 60 amino acids, usually greater than about 95
`amino acids, more usually greater than about 100 amino
`acids. Desirably, the H-rFv will be from about 110 to 125 10
`amino acids while the L-rFv will be from about 95 to 115
`amino acids.
`The amino acid compositions will vary widely, depending
`upon the particular idiotype involved. Usually there will be
`at least two cysteines separated by from about 60 to 75
`amino acids and joined by a disulfide bond to form cystine.
`The two chains will normally be substantial copies of
`idiotypes or the variable regions or the light and heavy
`chains of immunoglobulins, but in some situations it may be
`sufficient to have combinations of either the light or the
`heavy variable region chains.
`In many instances, it will be desirable to have one or both
`or the rFv chains labeled or bound to a support. Various
`labels may be employed, such as radioisotopes, fluorescers,
`or toxins. In other situatiollS, one or both of the chains 1nay 25
`be bound to an inert physiologically acceptable support,
`such as synthetic organic polymers, polysaccharides, natu(cid:173)
`rally occurring proteins, or other non-immunogenic sub(cid:173)
`stances.
`In some situations, it may be desirable to provide for 30
`covalent crosslinking of the two chains, which could involve
`providing for cysteine residues at the carboxyl termini. The
`chains will normally be prepared free of the constant
`regions, including or being free of all or a portion of the .T
`region. The D region will normally be included in the
`transcript of the H-rFv.
`For the most part only a relatively small percent of the
`total amino acids will vary from idiotype to idiotype in the
`rFv. Therefore, there will be areas providing a relatively 40
`constant framework and areas that will vary, namely, the
`hypervariable regions.
`The C-terminus region of the rPv will have a greater
`variety of sequences than the N-terminus and, based on the
`present strategy, can be further modified to permit variation
`from the naturally occurring heavy and light chains. A
`synthetic oligonucleotide can be employed to vary one or
`more amino acids in a hypervariable region.
`The preparation of the rFv employing hybrid DNA tech(cid:173)
`nology will now be described in greater detail.
`The preparation of the rFv will be divided into three parts:
`(1) isolation of appropriate DNA sequences; (2) introduction
`of the DNA sequences coding for the members of the rPv
`into an appropriate expression vector; and (3) expression
`and isolation of the mimetic variable regions of the light
`(L-rFv) and heavy (H-rFv) chains to provide the rFv.
`
`35
`
`4
`the last challenge, the spleen is removed and dissociated into
`single cells to be used for cell fusion to provide hybridomas.
`The immunogen will be the antigen of interest, or where
`a hapten, an antigenic conjugate of the hapten to an antigen.
`In order to prepare the hybridomas, the spleen cells are
`fused under conventional conditions employing a fusing
`agent, e.g. PEG6000, to a variety of inter- or intra- species
`myeloma cells, particularly mouse cells such as SP-2/0,
`NS-1, etc. and then suspended in HAT selective media. The
`surviving cells are then grown in microliter wells and
`immunologically assayed for production of antibodies to the
`determinant site(s) of interest.
`Assays for antibodies are well known in the art and may
`employ a variety or labeled antigens or haptens, where the
`labels are conveniently radioisotopes, lluorescers, enzymes,
`or the like. Other techniques may also be employed, such as
`sandwich techniques involving two antibodies, one bound to
`a support and the other being labeled. The cells from
`microliter wells scored as positive are cloned either by
`limiting dilution or cloning in soft agar. The resulting cloned
`cell lines are then propagated in an appropriate nutrient
`medium and, if necessary, may be stored frozen in liquid
`nitrogen.
`After selection of a particular cell line providing a mono(cid:173)
`clonal antibody of interest, the cells are expanded.
`Convenienlly, the cells may be grown to a density of about
`1x106 cellsiml in a 1 L culture. The cells are then harvested
`by centrifugation and lysed.
`In order to obtain the desired DNA sequence, one can look
`to either the gene expressing the variable region or the
`messenger RNA, which expresses the variable region. The
`difficulty with employing genomic DNA is in juxtaposing
`the sequences coding for the variable region, where the
`sequences are separated by introns. One must isolate the
`DNA fragment(s) containing the proper exons, excise the
`introns and then splice the exons in the proper order and
`orientation. For the most part, this will be difficult, so that
`the allernative technique employing the messenger RNA
`will be the method of choice.
`Where the messenger RNA is to be employed, the cells
`will be lysed under RNase inhibiting conditions. Themes(cid:173)
`senger RNA has the advantage that the mature messenger is
`free of introns, so that the sequence is continuous for the
`45 entire variable region. Difficulties with messenger RNA
`have been encountered, due to incomplete reverse transcrip(cid:173)
`tion but these difficulties can be minimized. The first step is
`to isolate the messenger RNA Conveniently, messenger
`RNA can be separated from other RNA because of its
`50 polyadenylation, employing an oligo-(dT) cellulose column.
`l'he mixture of messenger RNAs will be obtained free of
`other RNA The presence of messenger RNAs coding for the
`heavy and light chain polypeptides of the immunoglobulins
`may then be assayed by hybridization with DNA single
`55 strands of the appropriate genes. Conveniently, the
`sequences coding for the constant portion of the light and
`heavy chains may be used as probes, which sequences may
`be obtained from available sources (see, for example, Early
`and Hood, Genetic Engineering, Setlow and Hollaender eds.
`6U Vol. 3, Plenum Publishing Corp., New York (1981), pages
`157-188.)
`Whether the messenger RNA codes for the correct imnm(cid:173)
`noglobulin may be determined by in vitro translation
`employing a rabbit reticulocyte cell-free extract (Pelham and
`Jackson, I:urp. J. Diochem. (1976) 66:247-256). The result(cid:173)
`ing translation product may then be isolated by employing
`antibodies specific for one or more of the regions of the
`
`20
`
`I. Isolation of Appropriate DNA Sequences.
`In preparing the DNA sequences, a source of the genes
`encoding the variable region will be required. The variable
`regions may be derived from IgA, lgD, IgE, IgG or IgM,
`most commonly, from IgM and IgG. This can be achieved by
`in1mw1izing an appropriate vertebrate, normally a domestic
`animal, and most conveniently a mouse. The immunization
`may be carried out conventionally with one or more repeated 65
`injections of the immunogen into the host mammal, nor(cid:173)
`mally at two to three week intervals. Usually three days after
`
`Sanofi/Regeneron Ex. 1019, pg 579
`
`Merck Ex. 1019, pg 605
`
`

`
`5,840,545
`
`5
`chain of interest, for example, using rabbit anti( mouse IgG)
`where the chains are derived from mouse immunoglobulin.
`The immunoprecipitatc may be further analyzed by poly(cid:173)
`acrylamide gel electrophoresis, and the presence of com(cid:173)
`plexes cleterminecl by using racliotaggecl receptors for
`antigen-antibody complexes, such asS. aureus protein A, Rf
`factor, or the like. In addition, RNA blot hybridization can
`be employed to further insure that the correct messenger
`RNA is present.
`The crude mixture of mRNA sequences containing the
`desired mRNA sequences will be treated as follows. In order
`to enhance the probability that full length eDNA is obtained,
`the method of Okayama and Berg, Mol. Cell. Bioi. (19112)
`may be employed. Alternatively, the methods described by
`Efstradiadis and Villa-Komaroff (1979) in Genetic Engi- 15
`neering: Principles and Methods 1, Setlow and IIollaender,
`eels., New York, Plenum Press, pages 15-36, or Steinmetz ct
`al. (1981) Cell 24:125-134, may be employed. The first
`strand of eDNA is prepared employing a virus reverse
`transcriptase in the presence of prin1er. A second strand may
`then be prepared employing reverse transcriptase, the Kle(cid:173)
`now fragment of DNA polymerase I or T4 polymerase. If
`necessary, the resulting ds eDNA may then be treated with
`a single-strand-specific nuclease, such as S1 nuclease for
`removal of single stranded portions to result in ds eDNA, 25
`which may then be cloned.
`
`20
`
`6
`The host colonies, usually bacterial, which have DNA
`which hybridizes to either the light or heavy chain probes are
`picked and then grown in culture under selective pressure. In
`order to maintain selective pressure, it is desirable that the
`vector which is employed have biocidal, particularly
`antibiotic, resistance. After sufficient time for expansion of
`the host, the host cells arc harvested, conveniently by
`centrifugation. The hybrid plasmid DNA may then be iso(cid:173)
`lated by known procedures. (Gunsalus et al., J. Bacterial.
`10 (1979) 140:106-133).
`The isolated plasmid DNA is then characterized by
`restriction enzyme digestion and DNA sequence analysis.
`These analyses insure that the isolated eDNA clones com(cid:173)
`pletely encode the variable region and, optionally, the leader
`sequences for the light or heavy chain of the desired immu(cid:173)
`noglobulin. Furthermore, by having a restriction map of the
`variable regions and leader sequences, as well as the llank(cid:173)
`ing sequences, one can detem1ine the appropriate restriction
`sites for excising a DNA fragment which will allow for
`appropriate modification of the DNA sequence for insertion
`into a vector and expression of the polypeptide of interest.
`Where no unique restriction site is available at an appropri(cid:173)
`ate position in the flank-ing regions, partial digestion may be
`employed, with selection of fragments having the variable
`region and, optionally, the leader sequence intact. Where the
`5' and 3' flanking regions are too extended, these can be
`chewed back using llal31 to varying degrees by varying the
`period of digestion.
`Furthermore, by knowing the DNA sequence of the cod-
`30 ing strand in the region of the C-terminus of the heavy and
`light chain variable regions, a stop codon may be introduced
`at the C-terminus by the following procedme of in vitro
`mutagenesis. The eDNA is restricted with the appropriate
`enzyme(s) to provide a variable region coding segment with
`35 additional s· and 3' flanking sequences. This segment is
`purified, for example, by gel electrophoresis, gradient den(cid:173)
`sity centrifugation, etc. After isolating the desired segment,
`the two strands of the segment arc dissociated, conveniently
`by boiling. Alternatively, the undesired strand of the intact
`40 eDNA-plasmid clone may be nicked and digested.
`A synthetic, single-stranded DNA oligomer is prepared,
`conveniently by synthesis, which will have at least about 12
`nucleotides, more usually about 15 nucleotides, and will
`generally have fewer than about 50 nucleotides, usually
`45 fewer than 30 nucleotides, since a more extended oligomer
`is not required.
`Where heteroduplexing is involved, the non(cid:173)
`complementary nucleotides will usually be llanked by at
`least about three, more usually at least about six nucleotides
`50 complementary to the hybridized strand. The heteroduplex(cid:173)
`ing oligonudleotide will be complementary to the sequence
`at or about a significant juncture i.e. between the leader
`sequence and the variable region or the variable region and
`the constant region. The synthetic DNA oligomer will be
`55 complementary to the coding ("sense") strand of the
`variable-region sequence, but altered to encode a termina(cid:173)
`tion codon at the C-terminus of the variable region. That is,
`the oligomer will be complementary to the coding strand
`except at or about the amino acid which is involved at the
`6U juncture of the variable region and the D-, J- or C-regions of
`the light and heavy chains, particularly at or intermediate the
`U- or J -regions or intermediate the J-region, or at the J(cid:173)
`region and C-region juncture. It is intended that there will be
`some variation in the polypeptides which are prepared, so far
`65 as extending beyond the variable domains or not including
`all of the amino acids at the C -terminus of the variable
`region.
`
`II. Preparation of Genes Coding For I ,-rFv and H(cid:173)
`rFv and Introduction into an bxpression Vector For
`Amplification
`
`A wide variety of vectors may be employed for arnplifi(cid:173)
`cation or expression of the cis eDNA to produce the light and
`heavy chains of the immunoglobulin. A vector having an
`appropriate restriction site is digested with the appropriate
`endonuclease. The ds eDNA obtained from the reverse
`transcription of the mRNA may be modified by ligating
`linkers, treatment with terminal transferase or other tech(cid:173)
`niques to provide staggered (complementary) or blunt ended
`termini. The vectors may have one, two or more markers for
`selection of transformants. Desirably, the vector will have a
`unique restriction site in one of multiple markers, so that the
`transformants may be selected by the expression of one
`marker and the absence of expression of the other marker.
`Various markers may be employed, such as biocide
`resistance, complementation of an auxotroph, viral
`immtmity, or the like.
`After transforming an appropriate host with the cis eDNA
`prepared from the mRNA, e.g. E. coli, B. subtilis, S.
`cerevisiae, etc., in accordance with conventional ways, the
`transformants are plated and selected in accordance with the
`particular markers. The resulting colonies arc screened, by
`restriction electrophoretic pattern, hybridization to a labeled
`probe or by any other conventional means. See, for example,
`Hanahan and Meselson (19110), Gene 10:63-67. One proce(cid:173)
`dure employs colony hybridization, where the transformants
`are grown on a solid medium to produce colonies. Cells
`from the colonies are transferred to a nitrocellulose replica
`filter, the transferred cells incubated for further growth,
`lysed, dried and baked. The replica filter is then hybridized
`with appropriate radio-isotope labeled probes. conveniently,
`there are readily available probes for the determinant sites
`present in the constant regions of a variety of mammalian
`immunoglobulins. The colonies may be probed based on the
`nature of the particular immunoglobulin, as well as the
`different determinant sites, which may be present with the
`particular immunoglobulin.
`
`Sanofi/Regeneron Ex. 1019, pg 580
`
`Merck Ex. 1019, pg 606
`
`

`
`5,840,545
`
`20
`
`15
`
`7
`An excess amount of the oligomer is combined with the
`denatured strands of the restriction fragment under suffi(cid:173)
`cit:n!ly s!ringt:n! hybridization conditions. Thus, !ht: oligo(cid:173)
`mer specifically heteroduplexes to the complementary por(cid:173)
`tions of the coding strand, while providing one or more stop
`and/or nonsense codons to insure the termination of expres(cid:173)
`sion at the desired amino acid at the C-tcrminus.
`After sufficient time for hybridization at the desired level
`of stringency, sufficient amounts of !he four deoxynucle(cid:173)
`otides are added in conjunction with the Klenow fragment of 10
`DNA polymerase I. A strand complementary to the coding
`sequence of the variable-region and any 5'-fianking
`sequence is synthesized by enzymatic elongation of the
`primt:r rt:sul!ing in a st:qut:nct: complt:men!ary to !ht: strand
`to which the oligonucleotide is bound. The single-stranded
`DNA sequence on the coding strand located 3' to the region
`hybridized to the synthetic oligonucleotide is degraded by
`the 3'-5' exonuclease activity of the DNA polymerase. In
`this manner, ds eDNA is obtained which specifically codes
`for !ht: variabk-rt:gion and upstream llanking rt:gions asso-
`cia!t:d with !ht: light and heavy chains. Each of the heavy and
`light chains is encoded to tenninate expression at a prede(cid:173)
`termined codon in the V, D or J region.
`Tht: resulting he!erodupkxt:d blun!-t:ndt:d ds CDNAfrag(cid:173)
`ments are then employed for preparation of homoduplexed 25
`ds CDNA coding for the light and heavy variable regions
`with the stop codons at the desired sites. Conveniently, the
`blunt ended fragments arc modified as described previously,
`e.g. joined to linkers which code for restriction sites which
`are absent in the variable region sequences, or may he tailed 30
`e.g. polyG or polyC, or used directly for inSt:r!ion. With
`restriction site linkers, after insertion of the fragment into an
`appropriate vector having complementary termini, the frag(cid:173)
`ment can be recovered by restriction at the linker sites. The
`linkers arc joined to the coding sequences with an appro- 35
`priate ligase, e.g. T4 ligase, the resulting fragment restricted
`to provide cohesive ends, and the product annealed to the
`complemt:n!ary ends of a vector.
`At this stage, the vector which is employed provides for
`amplification and convenient isolation of transform ants hav- 40
`ing the variabk region coding st:quence insert. Numerous
`vt:ctors for amplification in bacteria or otht:r hosts exist such
`as pBR322, pSClOl, pRK290, 2 ,u-plasmid, etc. The hybrid
`plasmid containing the mismatched sequences will replicate
`in the host to generate two different plasmid molecules, one 45
`with the original sequence and one with the "tailored" or
`"site mutated" sequence derived from the synthetic oligo(cid:173)
`nudeo!idt:. Tht:refore, each !ransforman! colony is grown in
`small (approximatdy 2 ml) culture for plasmid isolation.
`The transform ants are grown, the plasmid DNA isolated 50
`in accordanct: with known proct:durt:s, and ust:d for a second
`cyck of transformation to provide inclividual clont:s rt:pli(cid:173)
`cating the tailored sequence. The clones may be screened by
`filter blot hybridization, probing with a labeled synthetic
`oligonucleotide which will include the synthetic oligonucle- 55
`otide employed in tailoring the variable region sequence, or
`other convenient technique. Thus, plasmids are obtained
`having ds eDNA llankt:d by appropriate rt:s!riction si!t:s and
`having a stop codon at a prt:detemiined site.
`Having now defined the 3'-terminus of the coding strand 6U
`or, al!ernativdy, the C-!erminus amino acid, the 5'-region or
`N-tem1inus of the polypeptide is now defined. Of course, tht:
`particular order in which the two termini are modified is
`primarily one of convenience, and can even be done
`simultaneously, where primer repair is used at the 5'-end of 65
`the coding strand in conjunction with site mutation at the
`3'-end.
`
`8
`Different strategies may be evolved, depending upon the
`nature of the host in which expression is to be obtained, and
`whether such host recognizes the leader sequence as a
`secretory signal for secretion of the polypeptide with con(cid:173)
`comitant removal of the leader sequence polypeptide. Where
`this opportunity is not available, the strategy will involve
`rt:moval of tht: kackr sequt:nce to provide a start codon at
`the 5'-terminus of the sequence of the coding strand coding
`for the variable region, which sequence can be inserted into
`an expression vector, so as to be under the control of a
`predetermined promoter and ribosomal start site.
`Based on tht: sequence of the leader region or tht:
`sequence coding for the N-terminus of the variable region,
`different oligonucleotides for homo- or heteroduplexing can
`be prepared.
`Whert: !ht: kadt:r st:quence is retained, primt:r repair is
`employt:d to remove tht: 5'-fianking sequt:nce of tht: coding
`strand. When the primer repair of the N-terminus is per(cid:173)
`formed simultaneously with the C-terminus mutagenesis,
`after treatment with the DNA polymerase, the resulting
`partial double stranded DNA will be treated with a 5'-3'(cid:173)
`single strand exonuclease to remove the 5'-fianking region as
`wdl as a ligaSt: to providt: for covaknt linking of !ht:
`rt:plicatt:d strand to tht: N -tt:rminus oligonucleotidt:.
`Where the leader sequence is to be removed, in vitro
`mutagenesis is employed to introduce an f-met codon at the
`N-terminus of the DNA sequence coding for the variable
`rt:gion.
`Alternative strategies may be employed for recovering the
`desired ds eDNA and performing the in vitro mutagenesis.
`If useful restriction sites are distant from the coding regions,
`the plasmid may be digested with the appropriate restriction
`endonuckase, followe